MCL-1 is a member of the B-Cell Lymphoma 2 (BCL-2) protein family that regulates cell death via mitochondrion-initiated apoptosis. Under normal conditions, antiapoptotic MCL-1 and its functional homologs BCL-2 and BCL-XL serve as prosurvival regulators that preserve mitochondrial integrity and protect against unwanted cell death. However, pathologic overexpression of these proteins subverts the natural death response and contributes to tumor initiation, progression, and resistance to chemotherapeutics. Prosurvival BCL-2 proteins have consequently become attractive targets for antitumor therapy. The most specific inhibitors of anti-apoptotic BCL-2 family proteins, ABT-737 and its orally bioavailable analog ABT-263, bind the hydrophobic binding groove of BCL-2 and BCL-XL to liberate trapped proapoptotic BCL-2 members. However, ABT-737/263 are unable to target MCL-1 due to its unique structure. Cancer cells with MCL-1 amplifications have been shown to be addicted to MCL-1 for survival, and MCL-1 is among the top 10 most frequently amplified genes in all human cancer types. Therefore, selective small molecule targeting of MCL-1 remains a promising unfulfilled goal for cancer therapy. In cancer cells, BIM is the most relevant activator BH3 that binds most tightly with MCL-1, yet current small molecule MCL-1 inhibitors have been reported to be able to displace tBID but not BIM from MCL-1. We have developed a cell-based high throughput screening strategy to identify small molecule inhibitors of the MCL-1-dependent survival pathway that focuses specifically on the MCL-1/BIM complex. This novel strategy enables the identification of small molecules that either directly release BIM by disrupting the binding of BIM to the hydrophobic binding pocket of MCL-1 or indirectly through targeting MCL-1 for degradation. We have conducted pilot screens using 1,900 compounds of the NCI Diversity Set and first 10,000 compounds of the ChemBridge DiverSet (50,080 compounds) and identified 17 lead compounds that triggered apoptosis in cancer cells that are addicted to MCL-1 for survival. Here we propose to use a top-down approach to (1) identify inhibitors of MCL-1 at any level of its regulation, elucidate their molecular mechanisms of action, and examine their functional capacities to sensitize cancer cell apoptosis; (2) screen the remaining 40,080 compounds of the ChemBridge DiverSet; (3) study the structure-activity relationship of MCL-1 lead compound inhibitors. We hypothesize that our screen will identify compounds that directly inhibit the hydrophobic binding groove of MCL-1, target MCL-1 for accelerated degradation, or induce proapoptotic BCL-2 proteins.